Chimeric Interaction of Nitrogenase‐Like Reductases with the MoFe Protein of Nitrogenase

Jasper, Jan; Ramos, José; Trncik, Christian; Jahn, Dieter; Einsle, Oliver; Layer, Gunhild; Moser, Jürgen

doi:10.1002/cbic.201900759

Cited by 6 publications

(9 citation statements)

References 53 publications

(72 reference statements)

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“…Intercomponent electron transfer is often facilitated via evolutionarily conserved protein docking faces [ 37 , 38 ]. Accordingly, chimeric carnitine monooxygenase enzymes consisting of individual subunits from E. coli (YeaX or YeaW) and A. baumannii (CntA or CntB) were reconstituted under conditions of the optimized assay.…”

Section: Resultsmentioning

confidence: 99%

Two-component carnitine monooxygenase from Escherichia coli: functional characterization, inhibition and mutagenesis of the molecular interface

et al. 2022

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Gut microbial production of trimethylamine (TMA) from L-carnitine is directly linked to cardiovascular disease. TMA formation is facilitated by carnitine monooxygenase which was proposed as a target for the development of new cardioprotective compounds. Therefore, the molecular understanding of the two-component Rieske-type enzyme from E. coli was intended. The redox cofactors of the reductase YeaX (FMN, plant-type [2Fe-2S] cluster) and of the oxygenase YeaW (Rieske-type [2Fe-2S] and mononuclear [Fe] center) were identified. Compounds meldonium and the garlic-derived molecule allicin were recently shown to suppress microbiota-dependent TMA formation. Based on two independent carnitine monooxygenase activity assays, enzyme inhibition by meldonium or allicin was demonstrated. Subsequently, the molecular interplay of the reductase YeaX and the oxygenase YeaW was addressed. Chimeric carnitine monooxygenase activity was efficiently reconstituted by combining YeaX (or YeaW) with the orthologous oxygenase CntA (or reductase CntB) from Acinetobacter baumannii. Partial conservation of the reductase/oxygenase docking interface was concluded. A structure guided mutagenesis approach was used to further investigate the interaction and electron transfer between YeaX and YeaW. Based on AlphaFold structure predictions, a total of 28 site-directed variants of YeaX and YeaW were kinetically analyzed. Functional relevance of YeaX residues Arg271, Lys313 and Asp320 was concluded. Concerning YeaW, a docking surface centered around residues Arg83, Lys104 and Lys117 was hypothesized. The presented results might contribute to the development of TMA-lowering strategies that could reduce the risk for cardiovascular disease.

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Section: Resultsmentioning

confidence: 99%

Two-component carnitine monooxygenase from Escherichia coli: functional characterization, inhibition and mutagenesis of the molecular interface

et al. 2022

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“…SHE) [25] . Our selection of electron donors/mediators was guided by the presumed structural and mechanistic similarities between BchL and nitrogenase's Fe protein [26] . To date the E 0 ’s of the [4Fe−4S] clusters of BchL and BchNB have not been determined; in the case of nitrogenase, the E 0 ’ of the Fe protein's [4Fe−4S] cluster is ∼ −0.3 V vs .…”

Section: Resultsmentioning

confidence: 99%

“…[25] Our selection of electron donors/mediators was guided by the presumed structural and mechanistic similarities between BchL ChemElectroChem and nitrogenase's Fe protein. [26] To date the E 0 's of the [4FeÀ 4S] clusters of BchL and BchNB have not been determined; in the case of nitrogenase, the E 0 ' of the Fe protein's [4FeÀ 4S] cluster is ~À 0.3 V vs. SHE and decreases to ~À 0.42 V upon the association of 2MgATP to the protein. [13] Further, methylviologen, triquat and Ti(III) citrate have all been found to support turnover by nitrogenase.…”

Section: Resultsmentioning

confidence: 99%

Alternative Electron Donors for the Nitrogenase‐like Dark‐Operative Protochlorophyllide Oxidoreductase (DPOR)

et al. 2022

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Sharing structural and mechanistic properties with the dinitrogen‐fixing enzyme nitrogenase, dark‐operative protochlorophyllide oxidoreductase (DPOR) participates in the biosynthesis of bacteriochlorophyll, subsequently enabling photosynthesis by photosynthetic bacteria. In contrast to the light‐dependent POR, electron transfer in DPOR is coupled to the hydrolysis of adenosine triphosphate for the two‐electron stereoselective reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), a coupling also employed by nitrogenases for the multielectron reduction of dinitrogen to ammonia. The importance of adenosine triphosphate hydrolysis is not fully understood for these enzymes, and electrochemistry is a valuable technique to regulate and/or monitor electron delivery to these enzymes. Here, we report three alternative electron donors that support protochlorophyllide reduction in the DPOR system, which could also enable electroenzymatic studies of DPOR. We demonstrate the turnover of DPOR within a spectroelectrochemical configuration, permitting the formation of Chlide to be followed and coupled to electron utilization by DPOR.

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“…Moreover, CfbC has been observed to form a weak complex with NifDK, the catalytic component of Mo-nitrogenase. Despite this interaction, CfbC + NifDK does not exhibit nitrogenase activity, indicating CfbC is unable to provide electrons to NifDK (34). CfbD is homologous to NifD but lacks a P-cluster or active site cofactor (e.g., FeMo-co).…”

Section: Discussionmentioning

confidence: 99%

The nitrogenase cofactor biogenesis enzyme NifB is essential for the viability of methanogens

Saini,

Dhamad,

Muniyasamy

et al. 2023

Preprint

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Dinitrogen (N2) is only bioavailable to select bacteria and archaea that possess the metalloenzyme nitrogenase, which reduces N2 to NH3 in a process called nitrogen fixation or diazotrophy. A long-term goal is to engineer diazotrophy into plants to decrease the use of nitrogen fertilizers, saving billions of dollars annually and greatly reducing nutrient pollution. This goal has not been realized, in part due to the inability to produce the nitrogenase metallocofactor within plants. Biogenesis of the cofactor requires NifB, a radical S-adenosy-L-methionine (SAM) enzyme that generates a precursor [8Fe-9S-C] cluster that matures into the final metallocofactor. Although maturation of nitrogenase is the only known function of NifB in bacteria, bioinformatic analyses reveal that NifB is conserved across methanogens, including those lacking nitrogenase, which suggests NifB functions outside of nitrogenase maturation. Indeed, several lines of evidence show that NifB is essential for viability of the model diazotroph, Methanosarcina acetivorans. First, CRISPRi repression was unable to abolish NifB production, whereas CRISPRi repression abolishes non-essential nitrogenase production. Second, unlike nitrogenase production, NifB production is not controlled by fixed nitrogen availability. Finally, nifB could not be deleted from M. acetivorans unless complemented in trans with nifB from other methanogens, including Methanothrix thermoacetophila, a species that lacks nitrogenase. Notably, M. thermoacetophila NifB supported diazotrophy in M. acetivorans, demonstrating that NifB from a non-diazotrophic methanogen produces the [8Fe-9S-C] cluster. Overall, these results link the metallocofactor biogenesis function of NifB to nitrogen fixation and methanogenesis, two processes of global importance.

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Chimeric Interaction of Nitrogenase‐Like Reductases with the MoFe Protein of Nitrogenase

Cited by 6 publications

References 53 publications

Two-component carnitine monooxygenase from Escherichia coli: functional characterization, inhibition and mutagenesis of the molecular interface

Two-component carnitine monooxygenase from Escherichia coli: functional characterization, inhibition and mutagenesis of the molecular interface

Alternative Electron Donors for the Nitrogenase‐like Dark‐Operative Protochlorophyllide Oxidoreductase (DPOR)

The nitrogenase cofactor biogenesis enzyme NifB is essential for the viability of methanogens

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